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从小麦根际土壤中分离到一株高产蛋白酶并对棉花枯萎病菌(Fusarium oxysporumf. sp. vasinfectum)具有拮抗作用的枯草芽孢杆菌(Bacillus subtilis)T2。采用硫酸铵分级沉淀、DEAE Sepharose Fast Flow、SP Sepharose Fast Flow离子交换层析、Sephadex G-75凝胶过滤层析,从T2发酵液中分离纯化出一种分子量约为29.0 kD的蛋白酶。该酶作用的最适温度为65℃,最适pH为8.0,在低于50℃、pH5.0~7.5范围内较稳定。Na+、K+、Pb2+、EDTA对酶有激活作用,Ca2+、Ba2+、Al3+、Zn2+、Mn2+、Hg2+、SDS对酶有部分抑制作用,而PMSF可完全抑制酶活,推测该酶是一种丝氨酸蛋白酶。抗菌活性显示,该酶对棉花枯萎病菌的孢子萌发、菌丝生长有明显抑制作用。
A Bacillus subtilis T2 strain with high protease activity and antagonistic activity against Fusarium oxysporumf. Sp. Vasinfectum was isolated from wheat rhizosphere soil. A protease with a molecular weight of 29.0 kD was isolated and purified from T2 fermentation broth by ammonium sulfate fractionation, DEAE Sepharose Fast Flow, SP Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography. The optimum temperature for the enzyme activity was 65 ° C and the optimum pH was 8.0, which was stable at pH 5.0 ~ 7.5 below 50 ° C. Enzymes were activated by Na +, K +, Pb2 + and EDTA. Ca2 +, Ba2 +, Al3 +, Zn2 +, Mn2 +, Hg2 + and SDS could partially inhibit the enzyme activity, while PMSF could completely inhibit the activity of the enzyme, suggesting that the enzyme is a serine protease. Antibacterial activity showed that the enzyme had a significant inhibitory effect on spore germination and mycelium growth of Fusarium oxysporum f.